Treatment of Megabacteria in Budgerigars by In-Water Medication with Soluble Amphotericin B.

Vetafarm Research Facility, Wagga Wagga, Australia

Abstract. Treatment of clinical megabacteriosis in Budgerigars was undertaken using a water soluble form of Amphotericin B. Treated birds were found to be free of detectable megabacteria following 10 days of medication. Megabacteriosis is recognised as a debilitating infection of Psittacines, especially the Budgerigar. Previously, flock treatments for megabacteriosis relied on performing crop gavage with a suspension of Amphotericin B twice daily for 10 days. The soluble form of Amphotericin B allows practical medication of large numbers of birds simultaneously.

Key Words: Amphotericin B, Budgerigar, Megabacteriosis
 
Introduction

Megabacteria have been described in the literature for several years. Various authors have examined the morphology, biochemistry, behaviour in culture, and therapeutics of the Megabacteria organism.^1-4 Debate is ongoing regarding the taxonomy of the organism.¹

Clinical veterinarians recognise two major forms of the disease known as Megabacteria Associated Disease (MAD) ^2,4 or Proventricular Ventricular Disease (PVD).¹ An acute disease is seen where large numbers of birds within a facility become acutely ill and a significant portion die within 12 - 24 hours. ⁵ The more commonly seen disease is a wasting disease associated with large numbers of the organisms colonising the proventriculus of the bird.^1,2

Treatment for MAD/PCD has involved a range of antibiotics and disinfectants administered orally either through crop gavage or in-water medication. Filippich trialled Amoxicillin, Metronidazole, Ketoconazole, Sodium carbonate-bicarbonate, Hydrochloric acid, Chloramine, and Chlorhexidine.³ None of the medications consistently eliminated megabacteria from the droppings of known positive Budgerigars. Filippich and Perry reported that treatment with Amphotericin B (Fungilin, Squibb) by crop gavage was successful in eliminating megabacteria from 28/30 birds treated.³ The dose rate for treatment with Amphotericin B has not been defined. Treatment regimes of 0.15 mL Fungilin ( 100 mg/mL Amphotericin B ) BID to 0.3 mL Fungilin 3 - 4 times daily have been suggested by Perry .⁴

Amphotericin is a polyene macrolide antifungal agent, which acts to bind the sterols (chiefly ergosterol) in fungal cell walls, altering permeability. It is reported to be inactive against bacteria, rickettsias, and viruses. If megabacteria are bacteria, which do not contain sterols in their cell wall, then the method of action of Amphotericin on megabacteria is unknown.¹

The Amphotericin molecule is a very large (mw = 924.1) which is practically insoluble in water, alcohol, or ether. There is little or no absorption of Amphotericin from the gastrointestinal tract. The toxic effects of Amphotericin are restricted to the intravenous administration of the colloidal form.⁶

The practical administration of Amphotericin B is the major obstacle to flock treatments for megabacteriosis. A water soluble complex of Amphotericin B was made by combining it with a cyclodextrin.

Cyclodextrins are degradation products of the action of cyclodextrin-glycosol-transferases on glucopyranoside chains.⁵ The resultant cyclodextrins have a large internal axial cavity. The outer surface is hydrophilic, while the internal structure is apolar. For practical purposes, cyclodextrins can be considered as empty cylindrical capsules of molecular size. When this cavity is filled with the molecule of another substance, it is known as an inclusion complex. Inclusion complexes have improved water solubility, improved bioavailability, enhanced stability, and elongated shelf life. ⁵

Materials and Method

An inclusion complex of gamma cyclodextrin and Amphotericin B was formed by adding an excess of Amphotericin B powder to a saturated solution of gamma cyclodextrin. Undissolved amphotericin was filtered out and the remaining solution freeze dried to form a yellow, amorphous powder. The resultant Amphotericin B/ Cyclodextrin complex (AMP/CD) had an Amphotericin content of 1.8 +/- 0.05% and was readily soluble in water.

Budgerigars with detectable megabacteria in their droppings were selected from a commercial breeding flock. Megabacteria positive birds were held in isolated breeding cabinets as a group and individual dropping screened by faecal Gram stain twice before selection for the trial. Only birds that were consistently positive to faecal Gram stain were selected for the trial.

The budgerigars were fed a commercial budgie mix and plain water.

Ten positive birds were selected and placed in a perspex hospital cage with the temperature maintained between 23 and 28⁰C. The birds had access to the commercial seed mix and medicated water.

Medicated water was provided by dissolving 5 grams of the AMP/CD in potable water giving a concentration of 0.9mg/mL Amphotericin B. A fresh solution was mixed and provided daily.

Individual dropping samples were obtained from each bird daily for 10 days by removing the birds from the hospital cage and placing in individual show cages without food or water. The cages were lined with clean disposable paper towel. As the birds passed a dropping it was collected and smeared on clean glass slides. Birds were returned to the hospital cage following collection of the dropping. Slides were Gram stained and examined as a group and results recorded as positive or negative for megabacteria.

The birds were monitored subjectively for changes in demeanour, activity, and the nature of their droppings.

At the end of the 10-day trial period, the birds were returned to the breeding cabinets. After 14 days in the cabinets individual samples from the birds were again collected, Gram stained, and examined.

Results

The results of faecal Gram staining indicated that the treated birds became faecal negative for megabacteria within 5 days of beginning the medicated water.

Samples designated +/- were difficult to interpret. There were structures present that, although of similar size to megabacteria, had poor staining and indistinct outline. It is presumed these were degenerating megabacteria organisms.

All follow-up samples, taken after 14 days back in the cabinets, were negative for the presence of megabacteria.

The birds under medication appeared to be more vocal and have increased activity after 3 - 5 days on the medication. A change in the nature of the droppings occurred during the trial. The droppings became smaller and darker with a more distinct urate portion.

Discussion

For effective flock treatment of megabacteriosis it was necessary to find a water soluble, stable form of Amphotericin B that could be easily administered by the birdkeeper.

The results of the trial indicate that the consumption of a soluble Amphotericin/Cyclodextrin complex by budgerigars eliminates megabacteria organisms from the droppings of budgerigars within 5 days. The droppings from the birds in the trial changed significantly in that they became more like the small black dropping of the normal budgerigar. The improvement in vocalisation and activity of the birds would suggest that the existing megabacterial infection was impacting on their general health and well being. The birds showed no evidence of ill effect from the period of medication.

The application of the AMP/CD complex was simple. The powder dissolved readily into tap water and the birds did not reject the medicated water.

Acknowledgement

This article is printed by kind permission of Vetafarm Research Facility, Wagga Wagga, Australia as part of the exchange of research information encouraged by the World Budgerigar Organisation (www.world-budgerigar.org) who supplied this article.

References

1 Bredhauer, B.G. (1996) Megabacteria and Proventricular/ventricular Disease in Australian Birds. Association of Avian Veterinarians, Australian Chapter Annual Conference Proceedings, Queensland, Australia.

2 Filippich, L. J. and M.G. Parker (1994).Megabacteria and Proventricular/Ventricular Disease in Psittacines and Passerines. Association of Avian Veterinarians Annual Conference, Reno, Nevada, USA.

3 Harrison, W. (1996) Megabacteriosis in Psittacine Birds. Association of Avian Veterinarians, Australian Chapter Annual Conference Proceedings, Queensland, Australia.

4 Perry, R.A. (1993) Megabacterium Associated Disease (M.A.D.). Avian Diagnostics, Proceedings 221, (1993) 13- 9. Post Graduate Committee in Veterinary Science.

5 Szetjtli, J. Medicinal Applications of Cyclodextrins. (1994) Medical Research Reviews 14(3): 353-386, John Wiley and Sons, Inc.

6 Martindale. The Extra Pharmocopoeia, 30^th edition (1993). pp. 315-318. The Pharmaceutical Press, London.